Placental Membrane Preparations and Methods of Making and Using Same for Regenerating Cartilage and Spinal Intervertebral Discs

ABSTRACT

A method for treating cartilage defects including providing a placental membrane preparation that comprises ground or minced placental membranes and optionally, a ground or minced cartilage and/or biocompatible glue, and introducing the preparation to a cartilage defect within a skeletal joint. The cartilage defect may comprise a hyaline cartilage defect, such as a chondral defect, or meniscal defect. The treatment may be provided in combination with other treatments such as marrow stimulation treatments and surgical repair treatments using sutures or other fixation techniques. The preparation promotes the regeneration of cartilage within the skeletal joint.

RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.61/993,114, filed on May 14, 2014 and titled “Placental MembranePreparations and Methods of Making and Using Same for RegeneratingCartilage and Spinal Intervertebral Discs,” and U.S. Utility patentapplication Ser. No. 14/712,156, filed on May 14, 2015 and titled“Placental Membrane Preparations and Methods of Making and Using Samefor Regenerating Cartilage and Spinal Intervertebral Discs,” the entirecontents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention is directed to placental membrane preparations.More particularly, the present invention is directed to placentalmembrane preparations and methods of making and using same forregenerating articular cartilage and spinal intervertebral discs.

BACKGROUND OF THE INVENTION

The placenta surrounds a fetus during gestation and is composed of,among other tissues, an inner amniotic layer that faces the fetus and agenerally inelastic outer shell, or chorion. The placenta anchors thefetus to the uterine wall, allowing nutrient uptake, waste elimination,and gas exchange to occur via the mother's blood supply. Additionally,the placenta protects the fetus from an immune response from the mother.From the placenta, an intact placental membrane comprising the amnionand chorion layers can be separated from the other tissues.

Clinicians have used intact placental membrane, comprising an amnion anda chorion layer, in medical procedures since as early as 1910 [Davis, J.S., John Hopkins Med. J. 15, 307 (1910)]. The amniotic membrane, whenseparated from the intact placental membrane, may also be used for itsbeneficial clinical properties [Niknejad H, et al. Eur Cell Mater 15,88-99 (2008)]. Certain characteristics of the placental membrane make itattractive for use by the medical community. These characteristicsinclude, but are not limited to, its anti-adhesive, anti-microbial, andanti-inflammatory properties; wound protection; ability to induceepithelialization; and pain reduction. [Mermet I, et al. Wound Repairand Regeneration, 15:459 (2007)].

Other uses for placental membrane include its use for scaffolding orproviding structure for the regrowth of cells and tissue. An importantadvantage of placental membrane in scaffolding is that the amnioncontains an epithelial layer. The epithelial cells derived from thislayer are similar to stem cells, allowing the cells to differentiateinto cells of the type that surrounds them. Multipotent cells similar tostem cells are also contained within the body of the amniotic membrane.Additionally, the amniotic membrane contains various growth and trophicfactors, such as epidermal, insulin-like, and fibroblast growth factors,as well as high concentrations of hyaluronic acid, that may bebeneficial to prevent scarring and inflammation and to support healing.Thus, placental membrane offers a wide variety of beneficial medicaluses.

Cell-based therapies have considerable potential for the repair andregeneration of tissues. The addition of a scaffold to these cell-basedtherapies has yielded improved outcomes [Krishnamurithy G, et al. JBiomed Mater Res Part A 99A, 500-506 (2011)]. Ideally, the material usedfor the scaffold will be biocompatible such that it provokes little tono immune response, biodegrades, and is available in sufficientquantities to be practical. Although the placental membrane has longbeen identified as a material potentially filling this role in theclinic, efforts have been limited to in vitro studies, impractical invivo techniques, or have yielded less than optimal outcomes.Furthermore, the conditions under which the scaffold is used may have adramatic effect on the therapeutic efficacy.

While a number of placental membrane products have been studied in theliterature or used clinically, these so far fall into two primarycategories. The first category involves the use of the intact membrane,be it fresh, dried, freeze-dried, cryopreserved, or preserved inglycerol or alcohol. In this formulation, the membrane is useful for anumber of purposes, but is not suitable for others, such as applicationsrequiring injection, or the filling of a space which does not conform tothe thin planar shape of the membrane itself.

The second category involves the grinding, pulverizing and/orhomogenizing of the membrane into small particles, which may then beresuspended in solution. Such techniques are described, for example, inU.S. patent application Ser. Nos. 11/528,902; 11/528,980; 11/529,658;and Ser. No. 11/535,924. This grinding may be done dry or wet, andtemperature during grinding may or may not be controlled, such as in thecase of cryogrinding. Products produced using this method are useful fora number of applications, and may be injected under appropriateconditions. However, they have several deficiencies for certainapplications. First, the cells contained in the placental membranes willbe destroyed during the grinding process. Second, proteins and growthfactors in the membrane may be leached out or lost during this process,including any subsequent washing or other treatment of the groundparticles. Indeed, the removal of potentially angiogenic factors such asgrowth factors may be an objective of this type of processing. Third,resuspension of these small particles in typical physiologic solutions,such as saline, results in a free-flowing fluid with low viscosity. Uponinjection or placement, this fluid may dissipate rather than remain inthe desired treatment location. Fourth, the resulting fragments may notbe large enough to permit cell engraftment and proliferation, if that isdesired.

However, amniotic membrane preparations have been shown to havesignificant beneficial bioactivity. Many of the cells contained in thesemembranes are multi- or pluri-potent. The membranes also contain a richsource of growth factors, as well as hyaluronic acid, collagen, andother factors which have been shown to support tissue healing. Amnioticmembrane has been shown to attract and stimulate the proliferation ofcells involved in tissue healing, such as mesenchymal stem cells andfibroblasts.

Articular cartilage, located on the articular ends of bones at jointsthroughout the body, is composed of hyaline cartilage and containsrelatively few chondrocytes that are embedded in extracellular matrixmaterials, such as type II collagen and proteoglycans [Moriya T, et al.J Orthop Sci 12, 265-273 (2007)]. Articular cartilage has a limitedability to self-repair, in part due to the avascular characteristics ofthe cartilage, which poses a significant challenge to treating jointinjuries or diseases. The repair of cartilage defects in humans cantherefore be a difficult endeavor, and multiple options exist for thesurgeon to approach this topic. The surgeon may choose to influence thedefect with microfracture, abrasion or other marrow stimulationtechniques which stimulate bleeding of the subchondral bone and thegeneration of a clot and ultimately a fibrocartilage patch which fillsthe defect. There are also options available that allow for the fillingof the defect with chondrocytes of variable sources, both of autograftand allograft origin.

A key advantage of marrow stimulation techniques over most otheravailable therapies is that marrow stimulation may be carried outarthroscopically using a relatively simple surgical technique, withminimal disruption to the joint and surrounding tissues. [Mithoefer, Ket. al. Chondral resurfacing of articular cartilage defects in the kneewith the microfracture technique. Surgical technique. J Bone Joint SurgAm. 2006 September; 88 Suppl 1 Pt 2:294-304]. The technique is alsocost-effective. Efforts have therefore been made to improve the outcomeof marrow stimulation techniques. Ground cartilage, either autograft orallograft (e.g. the product commercially marketed as BioCartilage) hasbeen proposed for this purpose. [Xing, L. et al. Microfracture combinedwith osteochondral paste implantation was more effective thanmicrofracture alone for full-thickness cartilage repair. Knee SurgSports Traumatol Arthrosc (2013) 21:1770-1776]. However the use ofautograft cartilage requires additional operative steps and donor sitemorbidity. The use of allograft cartilage alone has not been found tohave satisfactory results.

Current treatments, including cell-based therapies, have resulted in thegeneration of undesirable fibrocartilaginous tissue rather than hyalinecartilage [Diaz-Prado S M, et al. BIOMEDICAL ENGINEERING, TRENDS,RESEARCH, AND TECHNOLOGIES, pp. 193-216 (2011)]. As such, there remainsa significant clinical need for therapies capable of repairing damagedarticular cartilage that are capable of regenerating hyaline-likecartilage.

A similar need exists for solutions for the repair of meniscal defects.A meniscus is a crescent-shaped fibrocartilaginous structure that, incontrast to articular disks, only partly divides a joint cavity. Inhumans they are present in the knee, acromioclavicular,sternoclavicular, and temporomandibular joints. Generally, the term‘meniscus’ refers to the cartilage of the knee, either to the lateraland medial menisci. Both are cartilaginous tissues that providestructural integrity to the knee when it undergoes tension and torsion.They are concave on the top and flat on the bottom, articulating withthe tibia. They are attached to the small depressions (fossae) betweenthe condyles of the tibia (intercondyloid fossa), and towards the centerthey are unattached and their shape narrows to a thin shelf. The bloodflow of the meniscus is from the periphery to the central meniscus.Blood flow decreases with age and the central meniscus is avascular byadulthood leading to very poor healing rates. Meniscal defects arerepaired using sutures or other fixation approaches. Partialmeniscectomies are also commonly used. [Kon, E. et al. TissueEngineering for Total Meniscal Substitution: Animal Study in SheepModel—Results at 12 Months. Tissue Eng Part A. 2012 August;18(15-16):1573-82; Fiorentino G., et al. Easy and Safe All-Inside SutureTechnique for Posterior Horn Tears of Lateral Meniscus Using StandardAnteromedial and Anterolateral Portals. Arthroscopy Techniques, Vol 2,No 4 (November), 2013: pp e355-e359; Scotti C. et al. Meniscus RepairAnd Regeneration: Review On Current Methods And Research Potential.European Cells and Materials Vol. 26 2013, 150-170].

Another related problem involves the regeneration of the humanintervertebral disc. Intervertebral discs are fibrocartilaginous tissuesoccupying the space between vertebral bodies in the spine. They transmitforces from one vertebra to the next, while allowing spinal mobility.The structural properties of the disc are largely depending on itsability to attract and retain water. Proteoglycans in the disc exert anosmotic “swelling pressure” that resists compressive loads. Degenerationof the intervertebral disc is a physiologic process that ischaracteristic of aging in humans. With age, the disc undergoes avariety of changes, the most notable being a loss of proteoglycancontent resulting in reduced osmotic pressure and a reduction in discheight and ability to transmit loads. [Park, S. H., et. al.,Intervertebral Disk Tissue Engineering Using Biphasic Silk CompositeScaffolds. Tissue Eng. Part A, (2012) 18(5-6):447-458]. Discdegeneration is an important and direct cause of spinal conditions thataccount for most neck and back pain. As is the case with the relatedcartilage cells, components of the amniotic membrane may promote healingand recovery of the intervertebral disc and associated cells.

SUMMARY OF THE INVENTION

The present invention is directed to placental membrane preparations andmethods of making and using same. In some embodiments, the placentalmembranes may be ground or minced using techniques known in the art, andcombined with a biocompatible glue such as fibrin glue for cartilagerepair or disc regeneration. Such membranes may be further combined withground or minced autograft or allograft cartilage for implantation intoa defect.

In another embodiment, ground membrane particles may be injected intothe joint after marrow stimulation has been completed to stimulate thedevelopment of reparative cartilage. Such ground particles may becombined with prenatal stem cells, such as cells from the amniotic fluidor amniotic membrane, if desired. Such treatment may be repeated severaltimes at subsequent time periods if desired.

In another embodiment, the invention is directed to a method ofgenerating cartilage in vivo in a skeletal joint, the method includingconducting a marrow stimulation procedure, and then placing in thedefect a preparation of ground or minced amniotic membrane and abiocompatible glue. Ground or minced autograft or allograft cartilagemay also be included. The cartilage that is generated may comprise, inwhole or part, hyaline-like articular cartilage.

In another embodiment, the invention is directed to a method ofregenerating a damaged meniscus, the method including removing anydamaged meniscal tissue and filling the resulting void with an amnioticmembrane preparation with or without the addition of biocompatible glue.

In another embodiment, the invention is directed to a method ofregenerating a degenerated intervertebral disc, the method includinginserting minced amniotic membrane into the disc, with or without theaddition of biocompatible glue, and then closing any resulting openingin the disc using biocompatible glue or other closure means.

In certain embodiments, the invention is directed to a method ofgenerating cartilage in vivo in a skeletal joint comprising applying apreparation to a cartilage defect, wherein the preparation comprises aplacental membrane material selected from the group consisting of aground placental membrane, a minced placental membrane and combinationsthereof. The preparation may further comprise a processed cartilageselected from the group consisting of a ground cartilage, a mincedcartilage, a cartilage paste and combinations thereof. The processedcartilage may be selected from the group consisting of an autograftcartilage, an allograft cartilage and combinations thereof. In certainembodiments, the preparation comprises hyaluronic acid, saline or acombination thereof. The preparation may comprise the ground placentalmembrane wherein the ground placental membrane exhibits an averageparticle size of less than 0.1 mm. The preparation may comprise theminced placental membrane wherein the minced placental membrane exhibitsan average particle size within a range of 0.1 mm to 3 mm.

In certain embodiments, the placental membrane material comprises amniontissue containing organized amniotic extracellular matrix (ECM),amniotic tissue cells and growth factors contained within the ECM andamniotic tissue cells. The ECM may comprise amnion-derived collagen,fibronectin, laminin, proteoglycans and glycosaminoglycans. Theamnion-derived collagen may be derived from an epithelium layer, abasement membrane layer, a compact layer, a fibroblast layer, anintermediate layer and a spongy layer of the amnion tissue. Thepreparation may comprise amniotic fluid cells.

In certain embodiments, the preparation is applied by injection into thecartilage defect. The preparation may be introduced to the cartilagedefect using a minimally invasive procedure or through an arthroscopiccannula. The preparation may be injected into a joint capsule of theskeletal joint after a marrow stimulation procedure has been performedfor stimulating the development of a reparative cartilage in theskeletal joint.

In certain embodiments, the invention is directed to a method ofgenerating cartilage in vivo in a skeletal joint further comprising,following injecting the preparation into the joint capsule, evaluatingthe amount of in vivo cartilage generation within the skeletal joint,and based thereon, determining whether additional injections of thepreparation into the joint capsule are desired for accomplishing adesired amount of in vivo cartilage generation within the skeletaljoint.

In certain embodiments, the preparation comprises a biocompatible glue.In certain embodiments, the cartilage defect is a hyaline articularcartilage defect.

In certain embodiments, the invention is directed to a method ofgenerating cartilage in vivo in a skeletal joint further comprisingcausing blood to accumulate within the cartilage defect. In certainembodiments, the blood forms a clot within the cartilage defect. Incertain embodiments, the blood originates from subchondral bone locatedadjacent to the cartilage defect.

In certain embodiments, the invention is directed to a method ofgenerating cartilage in vivo in a skeletal joint further comprisingintroducing the preparation to the cartilage defect followingaccumulation of the blood within the cartilage defect. In certainembodiments, the method further comprises performing a marrowstimulation technique in the skeletal joint.

In certain embodiments, the preparation is configured to promote the invivo generation of hyaline cartilage within the skeletal joint. Incertain embodiments, the preparation is configured to promote the invivo generation of fibrocartilage within the skeletal joint. Thecartilage defect may be a meniscus cartilage defect. The preparation mayexclude in vitro cultured cells. The preparation may exclude in vitrocultured chondrocytes. The preparation may be configured to promote theregeneration of cartilage in the cartilage defect in the absence of invitro cultured cells.

In certain embodiments, the invention is directed to a method ofgenerating cartilage in vivo in a skeletal joint further comprisingremoving diseased cartilage from the skeletal joint thereby forming avoid into which the preparation is introduced. In certain embodiments,substantially all of a healthy cartilage in the skeletal joint remainsin the skeletal joint after the diseased cartilage is removed.

In certain embodiments, the preparation excludes a synthetic matrixmaterial. The preparation may be substantially free of chondrocytesimmediately prior to introduction to the cartilage defect.

In certain embodiments, the invention is directed to a method ofgenerating cartilage in vivo in a skeletal joint wherein a plurality ofcells contained within and native to the placental membrane materialchondrogenically differentiate in vivo within the cartilage defect. Theplurality of cells may comprise mesenchymal cells. The cartilage defectmay be an intervertebral disc defect.

In certain embodiments, the placental membrane material comprisessessile cells that are native to the placental membrane sheet. Incertain embodiments, the invention is directed to a method of generatingcartilage in vivo in a skeletal joint further comprising in vivochondrogenic differentiation of the sessile cells.

The placental membrane material may comprise intact placental membraneportions. The intact placental membrane portions may comprise sessileepithelial cells and sessile mesenchymal cells that are native to theplacental membrane intact placental membrane portions.

In certain embodiments, the invention is directed to a method ofgenerating cartilage in vivo in a skeletal joint comprising, identifyingdiseased cartilage in the skeletal joint, removing at least a portion ofthe diseased cartilage thereby forming a void, performing a marrowstimulation procedure within the skeletal joint and thereby causingblood to accumulate within the void, and inserting a preparation intothe void, the preparation comprising a minced placental membraneexhibiting an average placental membrane particle size within a range ofabout 0.1 mm to about 3 mm, the minced placental membrane comprisingamnion tissue containing organized amniotic extracellular matrix (ECM),sessile amnion tissue cells that are native to the placental membrane,growth factors contained within the ECM and sessile amnion tissue cells,and sessile amnion tissue-derived collagen, fibronectin, laminin,proteoglycans and glycosaminoglycans, wherein the sessile amniontissue-derived collagen is derived from an epithelium layer, a basementmembrane layer, a compact layer, a fibroblast layer, an intermediatelayer and a spongy layer of the sessile amnion tissue, wherein a portionof the sessile amnion tissue cells chondrogenically differentiate invivo within the void. The method may further comprise mincing an intactplacental membrane to produce the minced placental membrane.

In certain embodiments, the preparation comprises amniotic fluid cells.In certain embodiments, the preparation excludes in vitro culturedcells.

In certain embodiments, the invention is directed to a method ofgenerating cartilage in vivo in a skeletal joint comprising applying apreparation to cartilage in the skeletal joint, the preparationcomprising amniotic fluid cells and placental membrane portionscomprising sessile amnion tissue cells that are native to the placentalmembrane portions, and differentiating the sessile amnion tissue cellsinto chondrocytes in vivo within the skeletal joint. The method mayfurther comprise identifying diseased cartilage in the skeletal joint,removing at least a portion of the diseased cartilage thereby forming avoid, performing a marrow stimulation procedure within the skeletaljoint and thereby causing blood to accumulate within the void andinserting the preparation into the void. In certain embodiments, theplacental membrane portions have an average size within a range of about0.1 mm to about 3 mm.

A further understanding of the nature and advantages of the presentinvention will be realized by reference to the remaining portions of thespecification and the drawings of the present application.

DETAILED DESCRIPTION OF THE INVENTION

Before the present compositions, articles, devices, and/or methods aredisclosed and described, it is to be understood that they are notlimited to specific methods unless otherwise specified, or to particularreagents unless otherwise specified, and as such may vary. It is also tobe understood that the terminology as used herein is used only for thepurpose of describing particular embodiments and is not intended to belimiting.

This application references various publications. The disclosures ofthese publications, in their entireties, are hereby incorporated byreference into this application to describe more fully the state of theart to which this application pertains. The references disclosed arealso individually and specifically incorporated herein by reference formaterial contained within them that is discussed in the sentence inwhich the reference is relied on.

A. Definitions

In this specification, and in the claims that follow, reference is madeto a number of terms that shall be defined to have the followingmeanings:

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly indicates otherwise. Thus, forexample, reference to “a pharmaceutical carrier” includes mixtures oftwo or more such carriers, and the like.

As used herein, ranges can be expressed as from “about” one particularvalue, and/or to “about” another particular value. When such a range isexpressed, an embodiment includes from the one particular value and/orto the other particular value. Similarly, when values are expressed asapproximations, by the use of “about,” it will be understood that theparticular value forms another embodiment. It will be understood thatthe endpoints of each of the ranges are significant both in relation tothe other endpoint and independently of the other endpoint. It will alsobe also understood that there are a number of values disclosed herein,and that each value is also disclosed herein as “about” that particularvalue in addition to the value itself. For example, if the value “50” isdisclosed, then “about 50” is also disclosed. It is also understood thatwhen a value is disclosed that “less than or equal to” a value, thatvalues “greater than or equal to the value” and possible ranges betweenvalues are also disclosed, as understood by one skilled in the art. Forexample, if the value “50” is disclosed, then “less than or equal to 50”and “greater than or equal to 50” are also disclosed. It is alsounderstood that the throughout the application, data are provided indifferent formats, and it is understood that these data representendpoints and starting points as well as ranges for any combination ofthe data points. For example, if a particular data point “50” and aparticular data point “100” are disclosed, it is understood that greaterthan, greater than or equal to, less than, less than or equal to, andequal to 50 and 100 are considered disclosed as well as between 50 and100.

As used herein, “amniotic fluid cells” mean cells that have beenextracted, retrieved or derived from amniotic fluid from an amniotic sacof a pregnant female.

As used herein, “amniotic tissue” means amniotic fluid cells, placentalmembrane, amnion tissue or combinations thereof.

As used herein, the terms “optional” or “optionally” mean that thesubsequently described event or circumstance may or may not occur, andthat the description includes instances where said event or circumstanceoccurs and instances where it does not occur.

As used herein, the phrase “substantially all” refers to the maximumamount reasonably attainable by one skilled in the art.

As used herein, the term “particle size” means the particle size asdetermined, for example, by a laser scattering particle sizedistribution analyzer.

As used herein, the phrase “cartilage defect” refers to diseasedcartilage, a void created within cartilage by the removal of at least aportion of diseased cartilage, or a void created by cartilagedegeneration.

As used herein, the phrase “diseased cartilage” refers to cartilage thatis damaged, degenerating, inflamed, necrotic, or otherwise showingsymptoms thereof, such as pain, swelling, stiffness, and restraint ofmovement. Diseased cartilage may be diagnosed in several ways including,but not limited to, x-ray analysis, MRI analysis, or arthroscopy.

As used herein, the phrase “calcified cartilage” refers to the zone ofcartilage that connects articular cartilage to the underlyingsubchondral bone.

As used herein, the phrases “placental membrane” or “amnion tissue”refer to one or more layers of the placental membrane. For example,placental membrane or amnion tissue may refer to a placental membranecomprising both the amniotic and chorionic layers. In another example,placental membrane or amnion tissue may refer to a placental membrane inwhich the chorion has been removed. In another example, placentalmembrane or amnion tissue may refer to a placental membrane in which theepithelial layer has been removed.

As used herein, the phrase “subchondral bone” refers to bone underlyingcartilage. Subchondral bone may or may not be attached to the cartilage.

As used herein, the phrase “skeletal joint bone” refers to a bone incontact, or associated, with a skeletal joint. For example, a skeletaljoint bone associated with the knee joint may include the femur.

-   -   As used herein, the phrase “chondrogenic differentiation” refers        to the differentiation of one cell type into a chondrocyte or        chondrocyte-like cell. For example, mesenchymal stem cells may        undergo chondrogenic differentiation such that they        differentiate into chondrocytes.

As used herein, the phrase “prenatal stem cell” refers to a celloriginating from an embryonic or fetal mammalian organism and which isfound in or isolated from a prenatal sample. The term “mammalian” asused herein, encompasses any mammal, for instance a human. A “prenatalsample” is defined herein as a prenatal fluid or tissue. The term“prenatal fluid” is defined as mammalian third trimester amniotic fluid.A “prenatal tissue” is the fetal component of a mammalian placentaltissue, i.e., tissues originating predominantly from the fetus, forinstance placental membranes. The prenatal stem cells of the presentdisclosure specifically exclude stem cells isolated or collected from anadult source, i.e., any maternal components or maternal tissue presentin the mammalian placental membrane. A “stem cell” is a cell which hasthe potential to differentiate into multiple different cell types, andincludes both multipotent and pluripotent cells.

-   -   As used herein, the terms “treatment” or “treating” include any        desirable effect on the symptoms or pathology of a disease or        condition, and may include even minimal reductions in one or        more measurable markers of the disease or condition being        treated. “Treatment” does not necessarily indicate complete        eradication or cure of the disease or condition, or associated        symptoms thereof. The subject receiving this treatment is any        animal in need, including primates, in particular humans, and        other mammals including, but not limited to, equines, cattle,        swine, and sheep; and poultry and pets in general.

B. Making of the Placental Membrane Preparation

1. Placental Membrane Preparation.

The placental membrane preparation includes amnion tissue and,optionally, amniotic fluid cells. The amnion tissue component of theplacental membrane preparation is produced from placentas collected fromconsenting donors in accordance with the Current Good Tissue Practiceguidelines promulgated by the U.S. Food and Drug Administration. Inparticular, soon after the birth of a human infant via a Cesareansection delivery, the intact placenta is retrieved, and the placentalmembrane is dissected from the placenta. Afterwards, the placentalmembrane is cleaned of residual blood, placed in a bath of sterilesolution, stored on ice and shipped for processing. Once received by theprocessor, the placental membrane is rinsed to remove any remainingblood clots, and if desired, rinsed further in an antibiotic rinse[Diaz-Prado S M, et al. Cell Tissue Bank 11, 183-195 (2010)].

The antibiotic rinse may include, but is not limited to, theantibiotics: amikacin, aminoglycosides, amoxicillin, ampicillin,ansamycins, arsphenamine, azithromycin, azlocillin, aztreonam,bacitracin, capreomycin, carbacephem, carbapenems, carbenicillin,cefaclor, cefadroxil, cefalexin, cefalotin, cefamandole, cefazolin,cefdinir, cefditoren, cefepime, cefixime, cefoperazone, cefotaxime,cefoxitin, cefpodoxime, cefprozil, ceftaroline fosamil, ceftazidime,ceftibuten, ceftizoxime, ceftobiprole, ceftriaxone, cefuroxime,chloramphenicol, ciprofloxacin, clarithromycin, clindamycin,clofazimine, cloxacillin, colistin, cycloserine, dapsone, daptomycin,demeclocycline, di cloxacillin, dirithromycin, doripenem, doxycycline,enoxacin, ertapenem, erythromycin, ethambutol, ethionamide,flucloxacillin, fosfomycin, furazolidone, fusidic acid, gatifloxacin,geldanamycin, gentamicin, glycopeptides, grepafloxacin, herbimycin,imipenem or cilastatin, isoniazid, kanamycin, levofloxacin, lincomycin,lincosamides, linezolid, lipopeptide, lomefloxacin, loracarbef,macrolides, mafenide, meropenem, methicillin, metronidazole,mezlocillin, minocycline, monobactams, moxifloxacin, mupirocin,nafcillin, nalidixic acid, neomycin, netilmicin, nitrofurans,nitrofurantoin, norfloxacin, ofloxacin, oxacillin, oxytetracycline,paromomycin, penicillin G, penicillin V, piperacillin, platensimycin,polymyxin B, pyrazinamide, quinolones, quinupristin/dalfopristin,rifabutin, rifampicin or rifampin, rifapentine, rifaximin,roxithromycin, silver sulfadiazine, sparfloxacin, spectinomycin,spiramycin, streptomycin, sulfacetamide, sulfadiazine, sulfamethizole,sulfamethoxazole, sulfanilamide, sulfasalazine, sulfisoxazole,sulfonamidochrysoidine, teicoplanin, telavancin, telithromycin,temafloxacin, temocillin, tetracycline, thiamphenicol, ticarcillin,tigecycline, tinidazole, tobramycin, trimethoprim,trimethoprim-sulfamethoxazole (co-trimoxazole) (TMP-SMX), andtroleandomycin, trovafloxacin, or vancomycin.

The antibiotic rinse may also include, but is not limited to, theantimycotics: abafungin, albaconazole, amorolfin, amphotericin B,anidulafungin, bifonazole, butenafine, butoconazole, caspofungin,clotrimazole, econazole, fenticonazole, fluconazole, isavuconazole,isoconazole, itraconazole, ketoconazole, micafungin, miconazole,naftifine, nystatin, omoconazole, oxiconazole, posaconazole,ravuconazole, sertaconazole, sulconazole, terbinafine, terconazole,tioconazole, voriconazole, or other agents or compounds with one or moreanti-fungal characteristics.

The placental membrane may be processed to remove one or more particularlayers of the membrane. The chorion may be removed from the placentalmembrane by mechanical means well-known to those skilled in the art. Thechorion may be removed, for example, by carefully peeling the chorionfrom the remainder of the placental membrane using blunt dissection [JinC Z, et al. Tiss Eng 13, 693-702 (2007)]. Removal of the epitheliallayer from the placental membrane may be achieved using several methodswell-known to those skilled in the art. The epithelial layer may bepreserved or, if desired, may be removed by, for example, using trypsinto induce necrosis in the epithelial cells [Diaz-Prado S M, et al. CellTissue Bank 11, 183-195 (2010)]. Removal of the epithelial layer maycomprise, for example, treatment with 0.1%trypsin-ethylenediaminetetraacetic acid (EDTA) solution at 37° C. for 15minutes followed by physical removal using a cell scraper [Jin C Z, etal. Tiss Eng 13, 693-702 (2007)]. Preferably, the placental membraneutilized for the amnion tissue component of the placental membranepreparation is the amniotic membrane including the amniotic epithelialcell layers but excluding the chorion.

The placental membranes may be ground using techniques known in the art,and the resulting particles re-suspended in a fluid or dried. Suchprocessing may be carried out so as to preserve, to the extent possible,the protein content of the membrane, including growth factors.Preferably, grinding should be conducted under temperature controlledconditions, such as in a cryomill. Preferably such ground pieces oftissue should have a particle size of less than 1000 micrometers.Alternatively, the membranes may be minced using techniques known in theart, creating, e.g., small cubes of membrane tissue. Preferably suchminced pieces of tissue should have particle sizes ranging from 0.1 mmto 3 mm. Minced tissue particles may be square, rounded, oblong orirregular in shape.

The ground or minced placental membrane includes amnion tissuecontaining organized amniotic extracellular matrix (ECM), amniotictissue cells and growth factors contained within the ECM and amniotictissue cells. The ECM includes amnion-derived collagen, fibronectin,laminin, proteoglycans and glycosaminoglycans. The amnion-derivedcollagen may derived from an epithelium layer, a basement membranelayer, a compact layer, a fibroblast layer, an intermediate layer and aspongy layer of the amnion tissue.

The placental membrane preparation may be combined with prenatal stemcells if desired. For example the preparation may include amniotic fluidcells that are derived from amniotic fluid that is collected duringamniocentesis or scheduled C-section from consenting donors. Theamniotic fluid is spun thereby pelletizing the amniotic fluid cells. Theresulting amniotic fluid cells may be combined with ground placentalmembrane and cryopreserved in a solution containing approximately 5 to10% vol/vol Dimethyl Sulfoxide (DMSO) and 15 to 25% vol/vol protein,with the balance being crystalloids. Suitable dosages of the placentalmembrane preparation range from between about 0.25 ccs to about 5 ccs,depending on the amount of diseased or damaged tissue.

Minced or ground membrane particles may be freeze dried and sterilized,or stored in a cryopreservative or hypothermic storage solution allowingthe preservation of the viability of some membrane cells. A suitableground placental membrane preparation, which includes amniotic fluidcells, is sold by NuTech Medical, Inc. of Birmingham, Ala. under thename NuCel™.

The placental membrane preparation may include a processed cartilageselected from the group consisting of a ground cartilage, a mincedcartilage, a cartilage paste and combinations thereof. The processedcartilage may be an autograft cartilage, an allograft cartilage orcombinations thereof. When processed cartilage is added to a minced orground placental membrane preparation, the processed cartilage ispreferably provided in between a 3:1 and a 1:3 ratio by volume to theoriginal membrane preparation.

The placental membrane preparation may include hyaluronic acid, salineor a combination thereof. Hyaluronic acid and saline may be includedwith the placental membrane preparation when it is desired to inject thepreparation into a skeletal joint. When hyaluronic acid or saline isadded to a placental membrane preparation, the hyaluronic acid or salineis preferably provided in a 2:1 or 1:1 ratio by volume to the originalmembrane preparation.

The placental membrane preparation may include one or more biocompatibleglues. Biocompatible glues are natural polymeric materials that act asadhesives. Biocompatible glues may be formed synthetically frombiological monomers such as sugars and may consist of a variety ofsubstances, such as proteins and carbohydrates. Proteins such as gelatinand carbohydrates such as starch have been used as general-purpose gluesby man for many years. Preferably, the biocompatible glue is fibringlue, such as Tisseel. Fibrin is made up of fibrinogen (lyophilisedpooled human concentrate) and may also include thrombin (which may bereconstituted with calcium chloride).

C. Uses of the Placental Membrane Preparation

The embodiments of the placental membrane preparation, described herein,may be used to regenerate damaged or defective cartilage or disc tissue.Preferably, the embodiments of the placental membrane preparation,described herein, may be used to repair hyaline articular cartilage invivo. Alternatively, in cases of meniscal defects of the knee thepreparations may be used in conjunction with meniscal repair or partialmeniscectomy to repair defects in the meniscal cartilage. Alternatively,in cases of degenerated intervertebral discs the preparations may beused to restore disc height and function. The compositions and methodspertaining to the placental membrane preparation may be used in a numberof clinical conditions including, but not limited to, chondral defects,meniscal defects or tears, osteoarthritis, traumatic injury, such asrotational or compaction injuries, osteochondritis dessicans,pathological injury, age-related degeneration, and other defectsaffecting skeletal joints, in particular cartilage. Such techniques maybe used to address pathologies of the knee, shoulder, ankle, spine andother skeletal joints.

The membrane and glue preparation, with or without cartilage, may beplaced into a hyaline articular cartilage defect after a marrowstimulation procedure. Alternatively, the preparation may be placed in ameniscal defect prior to a defect repair via suture or other fixationtechniques, or may be placed into the defect created by a partialmeniscectomy. The preparation may be placed in a degeneratedintervertebral disc. The preparation may be placed in a minimallyinvasive manner via a syringe or arthroscopic cannula.

The ground membrane particles may be injected into the joint capsuleafter a marrow stimulation procedure has been completed to stimulate thedevelopment of reparative articular cartilage. Such injections may berepeated several times at subsequent time periods if desired. Theparticles may be combined with a biocompatible carrier such as saline orhyaluronic acid prior to injection.

Articular cartilage can be generated in vivo in a skeletal joint byconducting a marrow stimulation procedure, and then placing in thedefect a preparation of ground or minced amniotic membrane and abiocompatible glue. Ground or minced autograft or allograft cartilagemay also be included in the preparation. The method may be carried outin a minimally invasive manner using arthroscopic techniques.

Meniscal cartilage can be generated in vivo by conducting a partialmeniscectomy procedure, and then placing in the defect a preparation ofground or minced amniotic membrane, which may be mixed with or coveredby one or more biocompatible glues. Ground or minced autograft orallograft cartilage may also be included in the preparation. The methodmay be carried out in a minimally invasive manner using arthroscopictechniques.

A tear or defect in meniscal cartilage can be repaired in vivo in askeletal joint by placing in a meniscal tear or defect a preparation ofground or minced amniotic membrane with or without a biocompatible glue,followed by repairing of the tear or defect using suture or anotherfixation method. The method may be carried out in a minimally invasivemanner using arthroscopic techniques.

A degenerated intervertebral disc can be regenerated by inserting mincedamniotic membrane into the disc, with or without the addition ofbiocompatible glue, and then closing any resulting opening in the discusing biocompatible glue or other closure means.

The minced placental membrane in the placental membrane preparation maycontain living multi-potent prenatal cells if fresh or cryopreservedpreparations are used. The minced membrane may also act as a scaffold ormatrix for cell engraftment and in-growth. Thus, the minced membranesact as an integral matrix with cells intact in their normal location,i.e., sessile cells, and without culturing. The minced placentalmembranes also provide a reservoir of growth factors attracting incomingblood-born mesenchymal cells (MSCs), chondrocytes, and other reparativecells. In contrast, the ground placental membrane in the preparationincludes particles that may be too small to allow for cell in-growth.However, it is believed that the small particle sizes provides theplacental membrane preparation with a larger placental membrane surfacearea than surface area provided by minced placental membrane and thus,and may permit faster release of growth factors than the mincedmembrane. It is further believed that the small particle sizes allow forviable placental tissue cells to exist within the preparation.

Once applied to a cartilage defect or degenerated disc, the multi-potentamniotic cells, including those that are sessile and native to theplacental membrane sheet, may chondrogenically differentiate in vivo.The amniotic cells and the growth factors contained in the cells mayalso stimulate migration, differentiation, proliferation and matrixdeposition by the patient's own cells.

D. Example

The use of human amniotic allograft for treating osteo chondritisdissecans of the talar dome was observed.

Patients and Methods

Patients were selected from persons who had undergone arthroscopy withmicro-fracture technique for treatment of a talar dome lesion less than2 cm². Ankle scopes of 832 patients were reviewed. Three hundred andforty-five of those patients had lesions that were less than 2 cm² onthe talar dome. Patients were excluded from the study based on lack ofavailability of MRI scans, absence of solitary and isolated lesions,insufficient follow up times and whether patients had other majorsurgeries such as a peroneal tendon relocation or significant tibial,fibular, or talar exostectomy not done arthroscopically at the same timeas the lesion repair. To be included in the study, a patient'strans-chondral fracture (TCF) had to be reachable via arthroscopy andnot composed of a multi-planar shoulder lesion of the talus. Allpatients had modified American College of Foot and Ankle Surgeons(ACFAS) scores and visual analog scores (VAS) taken preoperatively andpostoperatively at 3 months, 12 months and 24 months. The patients hadMRI scans of lesions on their talar dome and did not have any othermajor surgeries at the time of surgically repairing the lesion.

After exclusions, the human amniotic allograft (HAA) group included 54patients with a talar dome lesion less than 2 cm2 whose treatmentincluded a human amniotic allograft to assist in healing andregeneration of cartilage. The control group consisted of 47 patientsthat had a talar dome lesion less than 2 cm² in size with no HAAallograft use. All patients had to complete four weeks of post-operativephysical therapy. Patients were not randomized or blinded to the use ofHAA.

Surgical Technique

Standard medial and lateral portals were used, standardized with auniform distraction technique. The ankle had an inspection, and then ageneralized synovectomy as indicated was done. As needed, a medial tolateral debridement and exostectomy of the anterior lip of the tibia wasperformed. Care was taken to assure that the tibial-talar interface hadno residual kissing lesion remaining. The talar dome lesion wasidentified and compared to x-ray and MRI size and location. Acircumferential debridement was performed to the subchondral level. Amicro-fracture awl standard technique was used to performmicro-fracturing of the lesion. When used, liquid form HAA was used andapplied directly to the lesion via needle technique and under directvisualization. Instruments were removed and portals were closed.

Human Amniotic Allograft (HAA) Information

The specific HAA material used on the patient population was 2 ccs of acryopreserved liquid form of amniotic allograft available from NuTechMedical, Inc. of Birmingham, Ala. and sold under the name NuCel®. NuCel®contains morselized amniotic membrane as well as other cells in theamniotic fluid of amniotic origin.

RESULTS

The average physical therapy for the control group pre-operatively was5.0 weeks and 5.7 weeks post-operative. The average for the treatmentgroup was 3.9 weeks preoperatively and 4.6 weeks postoperatively. Therewere no significant differences between the control and treatmentgroups' pre-operative and post-operative weeks in physical therapy(p=0.011 pre-operative, p=0.08 post-operative) as shown in Table 1.

TABLE 1 Patient Demographics with averages and p-values Physical TherapyVAS pain score (weeks) Post-op ACFAS Score Average Age Pre-op Post-opPre-op (24 months) Pre-op 3 month 12 month 24 month Graft 47.39 3.864.66 5.18 1.23 73.39 89.53 91.14 88.26 No Graft 46.01 5.09 5.74 5.022.48 74.39 84.7 86.19 83.93 p values Comparing* 0.3517 0.082 0.011 .353| 7E-6 .293 5 E-8 1 E-9 4 E-5 Graft** 1E-23 8 E-23 4 E-26 1 E-28 NoGraft** 2E-14 5 E-8 1 E-9 4 E-5 *p value comparing the category of thegraft group to the same category of non-graft group **p value comparingthe post-operative score to the pre-operative score.

The average VAS scores for the control and HAA groups were 5.0 and 5.2pre-operatively, respectively and 2.5 and 1.2 at 24 monthspost-operatively, respectively. There was no significant differencesbetween the pre-operative VAS scores (p=0.35) but a significantdifference in the post-operative VAS scores (p<0.001) was observed.There was also significance when comparing the pre and post-operativescores together for the control group and HAA group (p<0.001, p<0.001respectively), as shown in Table 1.

The ACFAS averages for the control and HAA groups were 74.4 and 73.4 forpreoperative, 84.7 and 89.5 at 3 months follow up, 86.2 and 91.1 at 12months follow up and 83.9 and 88.3 at 24 months follow up. The ACFASscores were not significant between the control and HAA pre-operativenumbers (p=0.293) but significant when comparing the control and HAAACFAS scores at 3 months, 12 months and 24 months post-operatively(p<0.001, p<0.001, p<0.001 respectively), as shown in Table 1. The totalpatient average width of the defect or bone edema from MRI scans was 1.9cm but 1.3 cm intra-operatively. There was an average difference of 0.6cm between the MRI and intra-operative size of the defect, with the MRIfindings showing larger or equal in all but one case (p<0.001).

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What is claimed is:
 1. A method of generating cartilage in vivo in askeletal joint comprising, applying a preparation to a cartilage defect,wherein the preparation comprises a placental membrane material selectedfrom the group consisting of a ground placental membrane, a mincedplacental membrane and combinations thereof.
 2. The method according toclaim 1 wherein the preparation comprises a processed cartilage selectedfrom the group consisting of a ground cartilage, a minced cartilage, acartilage paste and combinations thereof.
 3. The method according toclaim 2 wherein the processed cartilage is selected from the groupconsisting of an autograft cartilage, an allograft cartilage andcombinations thereof.
 4. The method according to claim 1 wherein thepreparation comprises hyaluronic acid, saline or a combination thereof.5. The method according to claim 1 wherein the preparation comprises abiocompatible glue.
 6. The method according to claim 1 wherein thepreparation comprises amniotic fluid cells.
 7. The method according toclaim 1 wherein the preparation excludes a synthetic matrix material, invitro cultured cells or a combination thereof.
 8. The method accordingto claim 1 wherein the placental membrane material comprises amniontissue comprising organized amniotic extracellular matrix (ECM),amniotic tissue cells and growth factors contained within the ECM andamniotic tissue cells.
 9. The method according to claim 8 wherein theECM comprises amnion-derived collagen, fibronectin, laminin,proteoglycans and glycosaminoglycans.
 10. The method according to claim9 wherein the amnion-derived collagen is derived from an epitheliumlayer, a basement membrane layer, a compact layer, a fibroblast layer,an intermediate layer and a spongy layer of the amnion tissue.
 11. Themethod according to claim 1 wherein the placental membrane materialcomprises intact placental membrane portions.
 12. The method accordingto claim 11 wherein the intact placental membrane portions comprisesessile epithelial cells and sessile mesenchymal cells that are nativeto the placental membrane intact placental membrane portions.
 13. Themethod according to claim 1 wherein the cartilage defect is selectedfrom the group consisting of a hyaline articular cartilage defect, ameniscus cartilage defect, and an intervertebral disc defect.
 14. Themethod according to claim 1 wherein the preparation is injected into ajoint capsule of the skeletal joint after performing a marrowstimulation procedure to stimulate the development of a reparativecartilage in the skeletal joint.
 15. The method according to claim 14further comprising, following injecting the preparation into the jointcapsule, evaluating the amount of in vivo cartilage generation withinthe skeletal joint, and based thereon, determining whether additionalinjections of the preparation into the joint capsule are desired foraccomplishing a desired amount of in vivo cartilage generation withinthe skeletal joint.
 16. The method according to claim 1 furthercomprising causing blood to accumulate within the cartilage defect. 17.The method according to claim 16, wherein the preparation is applied tothe cartilage defect following accumulation of the blood within thecartilage defect.
 18. The method according to claim 1 further comprisingperforming a marrow stimulation technique in the skeletal joint.
 19. Themethod according to claim 1 wherein the preparation promotes the in vivogeneration of hyaline cartilage within the skeletal joint.
 20. Themethod according to claim 1 wherein the preparation promotes the in vivogeneration of fibrocartilage within the skeletal joint.
 21. The methodaccording to claim 1 wherein the preparation promotes the regenerationof cartilage in the cartilage defect in the absence of in vitro culturedcells.
 22. The method according to claim 1 further comprising removingdiseased cartilage from the skeletal joint thereby forming a void intowhich the preparation is introduced.
 23. The method according to claim22 wherein substantially all of a healthy cartilage in the skeletaljoint remains in the skeletal joint after the diseased cartilage isremoved.
 24. The method according to claim 1 wherein a plurality ofcells contained within and native to the placental membrane materialchondrogenically differentiate in vivo within the cartilage defect. 25.The method according to claim 24 wherein the plurality of cells comprisemesenchymal cells.
 26. The method according to claim 1 wherein theplacental membrane material comprises sessile cells that are native tothe placental membrane sheet.
 27. The method according to claim 26further comprising in vivo chondrogenic differentiation of the sessilecells.
 28. A method of generating cartilage in vivo in a skeletal jointcomprising, identifying diseased cartilage in the skeletal joint,removing at least a portion of the diseased cartilage thereby forming avoid, performing a marrow stimulation procedure within the skeletaljoint and thereby causing blood to accumulate within the void, andinserting a preparation into the void, the preparation including aminced placental membrane exhibiting an average placental membraneparticle size within a range of about 0.1 mm to about 3 mm, the mincedplacental membrane comprising amnion tissue containing organizedamniotic extracellular matrix (ECM), sessile amnion tissue cells thatare native to the placental membrane, growth factors contained withinthe ECM and sessile amnion tissue cells, and sessile amniontissue-derived collagen, fibronectin, laminin, proteoglycans andglycosaminoglycans, wherein the sessile amnion tissue-derived collagenis derived from an epithelium layer, a basement membrane layer, acompact layer, a fibroblast layer, an intermediate layer and a spongylayer of the sessile amnion tissue, wherein a portion of the sessileamnion tissue cells chondrogenically differentiate in vivo within thevoid.
 29. The method according to claim 28 wherein the preparationcomprises amniotic fluid cells.
 30. The method according to claim 28wherein the preparation excludes in vitro cultured cells.
 31. The methodaccording to claim 28 further comprising mincing an intact placentalmembrane to produce the minced placental membrane.
 32. A method ofgenerating cartilage in vivo in a skeletal joint comprising, applying apreparation to cartilage in the skeletal joint, the preparationcomprising amniotic fluid cells and placental membrane portionscomprising sessile amnion tissue cells that are native to the placentalmembrane portions, and differentiating the sessile amnion tissue cellsinto chondrocytes in vivo within the skeletal joint.
 33. The methodaccording to claim 32 comprising identifying diseased cartilage in theskeletal joint, removing at least a portion of the diseased cartilagethereby forming a void, performing a marrow stimulation procedure withinthe skeletal joint and thereby causing blood to accumulate within thevoid and inserting the preparation into the void.